Secondary literature sources for H2B

BACKGROUND: Post-translational modifications of histones play importantroles in regulating nucleosome structure and gene transcription. It hasbeen shown that biotinylation of histone H4 at lysine-12 in histone H4(K12Bio-H4) is associated with repression of a number of genes. Wehypothesized that biotinylation modifies the physical structure ofnucleosomes, and that biotin-induced conformational changes contribute togene silencing associated with histone biotinylation.METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis we used atomicforce microscopy to directly analyze structures of nucleosomes formed withbiotin-modified and non-modified H4. The analysis of the AFM imagesrevealed a 13% increase in the length of DNA wrapped around the histonecore in nucleosomes with biotinylated H4. This statistically significant(p<0.001) difference between native and biotinylated nucleosomescorresponds to adding approximately 20 bp to the classical 147 bp lengthof nucleosomal DNA. CONCLUSIONS/SIGNIFICANCE: The increase in nucleosomalDNA length is predicted to stabilize the association of DNA with histonesand therefore to prevent nucleosomes from unwrapping. This provides amechanistic explanation for the gene silencing associated with K12Bio-H4.The proposed single-molecule AFM approach will be instrumental forstudying the effects of various epigenetic modifications of nucleosomes,in addition to biotinylation.

Charge state of the globular histone core controls stability of thenucleosome.

Biophys J. 2010; 99: 1577-85

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Presented here is a quantitative model of the wrapping and unwrapping ofthe DNA around the histone core of the nucleosome that suggests amechanism by which this transition can be controlled: alteration of thecharge state of the globular histone core. The mechanism is relevant toseveral classes of posttranslational modifications such as histoneacetylation and phosphorylation; several specific scenarios consistentwith recent in vivo experiments are considered. The model integrates adescription based on an idealized geometry with one based on the atomisticstructure of the nucleosome, and the model consistently accounts for boththe electrostatic and nonelectrostatic contributions to the nucleosomefree energy. Under physiological conditions, isolated nucleosomes arepredicted to be very stable (38 +/- 7 kcal/mol). However, a decrease inthe charge of the globular histone core by one unit charge, for exampledue to acetylation of a single lysine residue, can lead to a significantdecrease in the strength of association with its DNA. In contrast to theglobular histone core, comparable changes in the charge state of thehistone tail regions have relatively little effect on the nucleosome'sstability. The combination of high stability and sensitivity explains howthe nucleosome is able to satisfy the seemingly contradictory requirementsfor thermodynamic stability while allowing quick access to its DNAinformational content when needed by specific cellular processes such astranscription.

A genetic system to assess in vivo the functions of histones and histonemodifications in higher eukaryotes.

EMBO Rep. 2010; 11: 772-6

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Despite the fundamental role of canonical histones in nucleosomestructure, there is no experimental system for higher eukaryotes in whichbasic questions about histone function can be directly addressed. Wedeveloped a new genetic tool for Drosophila melanogaster in which thecanonical histone complement can be replaced with multiple copies ofexperimentally modified histone transgenes. This new histone-replacementsystem provides a well-defined and direct cellular assay system forhistone function with which to critically test models in chromatin biologydealing with chromatin assembly, variant histone functions and thebiological significance of distinct histone modifications in amulticellular organism.

Cytosine methylation on CpG dinucleotides is an essential epigeneticmodification in eukaryotes. How DNA methylation modulates nucleosomestructure and dynamics has been a long-standing question. We implemented asingle-molecule method to monitor the effects of DNA methylation on thestructure and dynamics of mononucleosomes. Our studies show that DNAmethylation induces a more compact and rigid nucleosome structure,providing a physical basis for how DNA methylation might contribute toregulating chromatin structure.

Nucleosomes compact and regulate access to DNA in the nucleus, and arecomposed of approximately 147 bases of DNA wrapped around a histoneoctamer. Here we report a genome-wide nucleosome positioning analysis ofArabidopsis thaliana using massively parallel sequencing ofmononucleosomes. By combining this data with profiles of DNA methylationat single base resolution, we identified 10-base periodicities in the DNAmethylation status of nucleosome-bound DNA and found that nucleosomal DNAwas more highly methylated than flanking DNA. These results indicate thatnucleosome positioning influences DNA methylation patterning throughoutthe genome and that DNA methyltransferases preferentially targetnucleosome-bound DNA. We also observed similar trends in human nucleosomalDNA, indicating that the relationships between nucleosomes and DNAmethyltransferases are conserved. Finally, as has been observed inanimals, nucleosomes were highly enriched on exons, and preferentiallypositioned at intron-exon and exon-intron boundaries. RNA polymerase II(Pol II) was also enriched on exons relative to introns, consistent withthe hypothesis that nucleosome positioning regulates Pol II processivity.DNA methylation is also enriched on exons, consistent with the targetingof DNA methylation to nucleosomes, and suggesting a role for DNAmethylation in exon definition.

Regulation of eukaryotic gene expression is far more complex than onemight have imagined 30 years ago. However, progress towards understandinggene regulatory mechanisms has been rapid and comprehensive, which hasmade the integration of detailed observations into broadly connectedconcepts a challenge. This review attempts to integrate the followingconcepts: (1) a well-defined organization of nucleosomes and modificationstates at most genes; (2) regulatory networks of sequence-specifictranscription factors; (3) chromatin remodeling coupled to promoterassembly of the general transcription factors and RNA polymerase II; and(4) phosphorylation states of RNA polymerase II coupled to chromatinmodification states during transcription. The wealth of new insightsarising from the tools of biochemistry, genomics, cell biology, andgenetics is providing a remarkable view into the mechanics of generegulation.

Two chromatin remodeling activities cooperate during activation of hormoneresponsive promoters.

PLoS Genet. 2009; 5: 1000567-1000567

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Steroid hormones regulate gene expression by interaction of theirreceptors with hormone responsive elements (HREs) and recruitment ofkinases, chromatin remodeling complexes, and coregulators to their targetpromoters. Here we show that in breast cancer cells the BAF, but not theclosely related PBAF complex, is required for progesterone induction ofseveral target genes including MMTV, where it catalyzes localizeddisplacement of histones H2A and H2B and subsequent NF1 binding. PCAF isalso needed for induction of progesterone target genes and acetylateshistone H3 at K14, an epigenetic mark that interacts with the BAF subunitsby anchoring the complex to chromatin. In the absence of PCAF, fullloading of target promoters with hormone receptors and BAF is precluded,and induction is compromised. Thus, activation of hormone-responsivepromoters requires cooperation of at least two chromatin remodelingactivities, BAF and PCAF.

Acetylation of histone H3 at the nucleosome dyad alters DNA-histonebinding.

J Biol Chem. 2009; 284: 23312-21

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Histone post-translational modifications are essential for regulating andfacilitating biological processes such as RNA transcription and DNArepair. Fifteen modifications are located in the DNA-histone dyadinterface and include the acetylation of H3-K115 (H3-K115Ac) and H3-K122(H3-K122Ac), but the functional consequences of these modifications areunknown. We have prepared semisynthetic histone H3 acetylated at Lys-115and/or Lys-122 by expressed protein ligation and incorporated them intosingle nucleosomes. Competitive reconstitution analysis demonstrated thatthe acetylation of H3-K115 and H3-K122 reduces the free energy of histoneoctamer binding. Restriction enzyme kinetic analysis suggests that thesehistone modifications do not alter DNA accessibility near the sites ofmodification. However, acetylation of H3-K122 increases the rate ofthermal repositioning. Remarkably, Lys --> Gln substitution mutations,which are used to mimic Lys acetylation, do not fully duplicate theeffects of the H3-K115Ac or H3-K122Ac modifications. Our results areconsistent with the conclusion that acetylation in the dyad interfacereduces DNA-histone interaction(s), which may facilitate nucleosomerepositioning and/or assembly/disassembly.

The packaging of eukaryotic DNA into chromatin sterically occludespolymerases, recombinases and repair enzymes. How chromatin structurechanges to allow their actions is unknown. We constructed definedfluorescently labeled trinucleosome arrays, allowing analysis of chromatinconformational dynamics via fluorescence resonance energy transfer (FRET).The arrays undergo reversible Mg2+-dependent folding similar to that oflonger arrays studied previously. We define two intermediateconformational states in the reversible folding of the nucleosome arraysand characterize the microscopic rate constants. Nucleosome arrays arehighly dynamic even when compact, undergoing conformational fluctuationson timescales in the second to microsecond range. Compact states of thearrays allow binding to DNA within the central nucleosome via siteexposure. Protein binding can also drive decompaction of the arrays. Thus,our results reveal multiple modes by which spontaneous chromatin fiberdynamics allow for the invasion and action of DNA-processing proteincomplexes.

We have previously reported that Major Histocompatibility Complex (MHC)class II can be induced by histone deacetylase inhibitors (HDACi) in theabsence of class II transactivator (CIITA). Here we characterized thehistone modifications associated with the CIITA-dependent (IFN-gammainduced) and -independent (HDACi induced) MHC class II expression. Wedemonstrate that both IFN-gamma and HDACi induced MHC class II expressionexhibited enhanced histone H3, H4 acetylation and H3K4me3 at the MHC classII promoter while H3K9me3 was decreased. In contrast, high levels ofH3K36me3 were detected at exons 3 and 5 but not at the promoter or thelocus control region (LCR). Interestingly, high levels of H3K79me2 wereonly detected at the promoter and exon 3 of the B cell lines while thelevel remained low and unchanged despite active MHC class II expressioninduced by either IFN-gamma or HDACi treatment. Constitutive expression ofthe CIITA protein by stable transfection of a CIITA deficient B cell linerestored the H3K79me2 to a level comparable to its cell of origin. Thisdata demonstrates that, although regulated by different pathways, bothIFN-gamma and HDACi treatments resulted in similar patterns of histonemodifications and that HDACi induce both histone methylation andacetylation. In addition, the different spatial distribution of the lysinemethylation markers along the gene suggests that these modifications playa distinctive role during different phases of the transcription process.

Gamma-H2AX in recognition and signaling of DNA double-strand breaks in thecontext of chromatin.

Nucleic Acids Res. 2008; 36: 5678-94

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DNA double-strand breaks (DSBs) are extremely dangerous lesions withsevere consequences for cell survival and the maintenance of genomicstability. In higher eukaryotic cells, DSBs in chromatin promptly initiatethe phosphorylation of the histone H2A variant, H2AX, at Serine 139 togenerate gamma-H2AX. This phosphorylation event requires the activation ofthe phosphatidylinositol-3-OH-kinase-like family of protein kinases,DNA-PKcs, ATM, and ATR, and serves as a landing pad for the accumulationand retention of the central components of the signaling cascade initiatedby DNA damage. Regions in chromatin with gamma-H2AX are convenientlydetected by immunofluorescence microscopy and serve as beacons of DSBs.This has allowed the development of an assay that has proved particularlyuseful in the molecular analysis of the processing of DSBs. Here, we firstreview the role of gamma-H2AX in DNA damage response in the context ofchromatin and discuss subsequently the use of this modification as asurrogate marker for mechanistic studies of DSB induction and processing.We conclude with a critical analysis of the strengths and weaknesses ofthe approach and present some interesting applications of the resultingmethodology.

Using the massively parallel technique of sequencing by oligonucleotideligation and detection (SOLiD; Applied Biosystems), we have assessed thein vivo positions of more than 44 million putative nucleosome cores in themulticellular genetic model organism Caenorhabditis elegans. Theseanalyses provide a global view of the chromatin architecture of amulticellular animal at extremely high density and resolution. While weobserve some degree of reproducible positioning throughout the genome inour mixed stage population of animals, we note that the major chromatinfeature in the worm is a diversity of allowed nucleosome positions at thevast majority of individual loci. While absolute positioning ofnucleosomes can vary substantially, relative positioning of nucleosomes(in a repeated array structure likely to be maintained at least in part bysteric constraints) appears to be a significant property of chromatinstructure. The high density of nucleosomal reads enabled a substantialextension of previous analysis describing the usage of individualoligonucleotide sequences along the span of the nucleosome core andlinker. We release this data set, via the UCSC Genome Browser, as aresource for the high-resolution analysis of chromatin conformation andDNA accessibility at individual loci within the C. elegans genome.

Biological complexes are typically multisubunit in nature and theprocesses in which they participate often involve protein compositionalchanges in themselves and/or their target substrates. Being able toidentify more than one type of protein in complex samples and to trackcompositional changes during processes would thus be very useful. Towardthis goal, we describe here a single-molecule technique that cansimultaneously identify two types of proteins in compositionally complexsamples. It is an adaptation of the recently developed atomic forcemicroscopy (AFM) recognition imaging technique but involves the tetheringof two different types of antibodies to the AFM tip and sequentialblocking with appropriate antigenic peptides to distinguish therecognition from each antibody. The approach is shown to be capable ofsimultaneously identifying in a single AFM image two specific components,BRG1 and beta-actin, of the human Swi-Snf ATP-dependent nucleosomeremodeling complex and two types of histones, H2A and H3, in chromatinsamples.

Choreography for nucleosomes: the conformational freedom of thenucleosomal filament and its limitations.

Nucleic Acids Res. 2007; 35: 106-106

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Eukaryotic DNA is organized into nucleosomes by coiling around coreparticles of histones, forming a nucleosomal filament. The significancefor the conformation of the filament of the DNA entry/exit angle (alpha)at the nucleosome, the angle of rotation (beta) of nucleosomes aroundtheir interconnecting DNA (linker DNA) and the length of the linker DNA,has been studied by means of wire models with straight linkers. It isshown that variations in alpha and beta endow the filament with anoutstanding conformational freedom when alpha is increased beyond 60-90degrees, owing to the ability of the filament to change between forwardright-handed and backward left-handed coiling. A wealth of differenthelical and looped conformations are formed in response to repeated betasequences, and helical conformations are shown to be able to contract to ahigh density and to associate pairwise into different types of doublefibers. Filaments with random beta sequences are characterized byrelatively stable loop clusters connected by segments of higherflexibility. Displacement of core particles along the DNA in such fibers,combined with limited twisting of the linkers, can generate the betasequence necessary for compaction into a regular helix, thus providing amodel for heterochromatinization.

Nucleosome positioning signals embedded within the DNA sequence have thepotential to influence the detailed structure of the higher-orderchromatin fibre. In two previous studies of long stretches of DNA,encompassing the chicken beta-globin and ovine beta-lactoglobulin genes,respectively, we mapped the relative affinity of every site for the corehistone octamer. In both cases a periodic arrangement of the in vitropositioning sites suggests that they might influence the folding of anucleosome chain into higher-order structure; this hypothesis was borneout in the case of the beta-lactoglobulin gene, where the distribution ofthe in vitro positioning sites is related to the positions nucleosomesactually occupy in sheep liver cells. Here, we have exploited the in vitronucleosome positioning datasets to simulate nucleosomal organisation usingin silico approaches. We use the high-resolution, quantitative positioningmaps to define a one-dimensional positioning energy lattice, which can bepopulated with a defined number of nucleosomes. Monte Carlo techniques areemployed to simulate the behaviour of the model at equilibrium to producea set of configurations, which provide a probability-based occupancy map.Employing a variety of techniques we show that the occupancy maps are asensitive function of the histone octamer density (nucleosome repeatlength) and find that a minimal change in this property can producedramatic localised changes in structure. Although simulations generallygive rise to regular periodic nucleosomal arrangements, they often showoctamer density-dependent discontinuities, which tend to co-localise withsequences that adopt distinctive chromatin structure in vivo. Furthermore,the overall organisation of simulated chromatin structures are moreclosely related to the situation in vivo than is the original in vitropositioning data, particularly at a nucleosome density corresponding tothe in vivo state. Although our model is simplified, we argue that itprovides a unique insight into the influence that DNA sequence can have indetermining chromatin structure and could serve as a useful basis for theincorporation of other parameters.

We describe a new mesoscopic model of oligonucleosomes that incorporatesflexible histone tails. The nucleosome cores are modeled using thediscrete surface-charge optimization model, which treats the nucleosome asan electrostatic surface represented by hundreds of point charges; thelinker DNAs are treated using a discrete elastic chain model; and thehistone tails are modeled using a bead/chain hydrodynamic approach aschains of connected beads where each bead represents five proteinresidues. Appropriate charges and force fields are assigned to eachhistone chain so as to reproduce the electrostatic potential, structure,and dynamics of the corresponding atomistic histone tails at differentsalt conditions. The dynamics of resulting oligonucleosomes at differentsizes and varying salt concentrations are simulated by Brownian dynamicswith complete hydrodynamic interactions. The analyses demonstrate that thenew mesoscopic model reproduces experimental results better than itspredecessors, which modeled histone tails as rigid entities. Inparticular, our model with flexible histone tails: correctly accounts forsalt-dependent conformational changes in the histone tails; yields theexperimentally obtained values of histone-tail mediated core/coreattraction energies; and considers the partial shielding of electrostaticrepulsion between DNA linkers as a result of the spatial distribution ofhistone tails. These effects are crucial for regulating chromatinstructure but are absent or improperly treated in models with rigidhistone tails. The development of this model of oligonucleosomes thusopens new avenues for studying the role of histone tails and theirvariants in mediating gene expression through modulation of chromatinstructure.

Although numerous experiments indicate that the chromatin fiber displayssalt-dependent conformations, the associated molecular mechanism remainsunclear. Here, we apply an irregular Discrete Surface Charge Optimization(DiSCO) model of the nucleosome with all histone tails incorporated todescribe by Monte Carlo simulations salt-dependent rearrangements of anucleosomal array with 12 nucleosomes. The ensemble of nucleosomal arrayconformations display salt-dependent condensation in good agreement withhydrodynamic measurements and suggest that the array adopts highlyirregular 3D zig-zag conformations at high (physiological) saltconcentrations and transitions into the extended "beads-on-a-string"conformation at low salt. Energy analyses indicate that the repulsionamong linker DNA leads to this extended form, whereas internucleosomeattraction drives the folding at high salt. The balance between these twocontributions determines the salt-dependent condensation. Importantly, theinternucleosome and linker DNA-nucleosome attractions require histonetails; we find that the H3 tails, in particular, are crucial forstabilizing the moderately folded fiber at physiological monovalent salt.

Structure of the conserved core of the yeast Dot1p, a nucleosomal histoneH3 lysine 79 methyltransferase.

J Biol Chem. 2004; 279: 43296-306

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Methylation of Lys79 on histone H3 by Dot1p is important for genesilencing. The elongated structure of the conserved core of yeast Dot1pcontains an N-terminal helical domain and a seven-stranded catalyticdomain that harbors the binding site for the methyl-donor and an activesite pocket sided with conserved hydrophobic residues. TheS-adenosyl-L-homocysteine exhibits an extended conformation distinct fromthe folded conformation observed in structures of SET domain histonelysine methyltransferases. A catalytic asparagine (Asn479), located at thebottom of the active site pocket, suggests a mechanism similar to thatemployed for amino methylation in DNA and protein glutamine methylation.The acidic, concave cleft between the two domains contains two basicresidue binding pockets that could accommodate the outwardly protrudingbasic side chains around Lys79 of histone H3 on the disk-like nucleosomesurface. Biochemical studies suggest that recombinant Dot1 proteins areactive on recombinant nucleosomes, free of any modifications.

Intra- and inter-nucleosomal protein-DNA interactions of the core histonetail domains in a model system.

J Biol Chem. 2003; 278: 24217-24

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The core histone tail domains are key regulators of eukaryotic chromatinstructure and function and alterations in the tail-directed folding ofchromatin fibers and higher order structures are the probable outcome ofmuch of the post-translational modifications occurring in these domains.The functions of the tail domains are likely to involve complex intra- andinter-nucleosomal histone-DNA interactions, yet little is known abouteither the structures or interactions of these domains. Here we introducea method for examining inter-nucleosome interactions of the tail domainsin a model dinucleosome and determine the propensity of each of the fourN-terminal tail domains to mediate such interactions in this system. Usinga strong nucleosome "positioning" sequence, we reconstituted a nucleosomecontaining a single histone site specifically modified with aphotoinducible cross-linker within the histone tail domain, and a secondnucleosome containing a radiolabeled DNA template. These two nucleosomeswere then ligated together and cross-linking induced by brief UVirradiation under various solution conditions. After cross-linking, thetwo templates were again separated so that cross-linking representinginter-nucleosomal histone-DNA interactions could be unambiguouslydistinguished from intra-nucleosomal cross-links. Our results show thatthe N-terminal tails of H2A and H2B, but not of H3 and H4, makeinternucleosomal histone-DNA interactions within the dinucleosome. Therelative extent of intra- to inter-nucleosome interactions was notstrongly dependent on ionic strength. Additionally, we find that bindingof a linker histone to the dinucleosome increased the association of theH3 and H4 tails with the linker DNA region.

Is a small number of charge neutralizations sufficient to bend nucleosomecore DNA onto its superhelical ramp?

J Am Chem Soc. 2003; 125: 15087-92

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X-ray diffraction structures of the nucleosome core particle along with avariety of experiments are consistent with the idea that an importantsource of the free energy holding DNA to the superhelical ramp on thehistone octamer surface is obtained from a relatively small amount ofelectrostatic neutralization of the DNA phosphate charge by positivelycharged histone groups, especially arginine residues. Here we present atheoretical analysis of a simple model that emphasizes the competitionbetween the high degree of bending of the stiff DNA molecule required forits tight curvature on the histone octamer and the neutralization of theDNA phosphate charge by basic histone residues. Our calculation accountsfor the strong influence of condensed counterions on the electrostaticinteractions. We find that the minimum amount of free energy required tobend DNA into axial conformity with the superhelical ramp at physiologicalsalt concentration can be provided by a scant 6% neutralization of thephosphate charge, in close correspondence to the stoichiometricneutralization of phosphate charge by the arginine side chain thatintrudes into the inward-facing minor groove of each DNA double helicalturn.

Sequence-dependent dynamics of duplex DNA: the applicability of adinucleotide model.

Biophys J. 2002; 83: 3446-59

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The short-time (submicrosecond) bending dynamics of duplex DNA weremeasured to determine the effect of sequence on dynamics. All measurementswere obtained from a single site on duplex DNA, using a single,site-specific modified base containing a rigidly tethered, electronparamagnetic resonance active spin probe. The observed dynamics areinterpreted in terms of single-step sequence-dependent bending forceconstants, determined from the mean squared amplitude of bending relativeto the end-to-end vector using the modified weakly bending rod model. Thebending dynamics at a single site are a function of the sequence of thenucleotides constituting the duplex DNA. We developed and examined severaldinucleotide-based models for flexibility. The models indicate that thedominant feature of the dynamics is best explained in terms of purine- andpyrimidine-type steps, although distinction is made among all 10 uniquesteps: It was found that purine-purine steps (which are the same aspyrimidine-pyrimidine steps) were near average in flexibility, but thepyrimidine-purine steps (5' to 3') were nearly twice as flexible, whereaspurine-pyrimidine steps were more than half as flexible as average DNA.Therefore, the range of stepwise flexibility is approximately fourfold andis characterized by both the type of base pair step (pyrimidine/purinecombination) and the identity of the bases within the pair (G, A, T, orC). All of the four models considered here underscore the complexity ofthe dependence of dynamics on DNA sequence with certain sequences notsatisfactorily explainable in terms of any dinucleotide model. Thesefindings provide a quantitative basis for interpreting the dynamics andkinetics of DNA-sequence-dependent biological processes, including proteinrecognition and chromatin packaging.

Purifying selection and birth-and-death evolution in the histone H4 genefamily.

Mol Biol Evol. 2002; 19: 689-97

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Histones are small basic proteins encoded by a multigene family and areresponsible for the nucleosomal organization of chromatin in eukaryotes.Because of the high degree of protein sequence conservation, it isgenerally believed that histone genes are subject to concerted evolution.However, purifying selection can also generate a high degree of sequencehomogeneity. In this study, we examined the long-term evolution of histoneH4 genes to determine whether concerted evolution or purifying selectionwas the major factor for maintaining sequence homogeneity. We analyzed theproportion (p(S)) of synonymous nucleotide differences between the H4genes from 59 species of fungi, plants, animals, and protists and foundthat p(S) is generally very high and often close to the saturation level(p(S) ranging from 0.3 to 0.6) even though protein sequences are virtuallyidentical for all H4 genes. A small proportion of genes showed a low levelof p(S) values, but this appeared to be caused by recent gene duplication.Our findings suggest that the members of this gene family evolve accordingto the birth-and-death model of evolution under strong purifyingselection. Using histone-like genes in archaebacteria as outgroups, wealso showed that H1, H2A, H2B, H3, and H4 histone genes in eukaryotes formseparate clusters and that these classes of genes diverged nearly at thesame time, before the eukaryotic kingdoms diverged.

Mutations in Drosophila ISWI, a member of the SWI2/SNF2 family ofchromatin remodeling ATPases, alter the global architecture of the male Xchromosome. The transcription of genes on this chromosome is increased2-fold relative to females due to dosage compensation, a process involvingthe acetylation of histone H4 at lysine 16 (H4K16). Here we show thatblocking H4K16 acetylation suppresses the X chromosome defects resultingfrom loss of ISWI function in males. In contrast, the forced acetylationof H4K16 in ISWI mutant females causes X chromosome defectsindistinguishable from those seen in ISWI mutant males. Increasedexpression of MOF, the histone acetyltransferase that acetylates H4K16,strongly enhances phenotypes resulting from the partial loss of ISWIfunction. Peptide competition assays revealed that H4K16 acetylationreduces the ability of ISWI to interact productively with its substrate.These findings suggest that H4K16 acetylation directly counteractschromatin compaction mediated by the ISWI ATPase.

Kinetics of core histones in living human cells: little exchange of H3 andH4 and some rapid exchange of H2B.

J Cell Biol. 2001; 153: 1341-53

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Histones H2A and H2B form part of the same nucleosomal structure as H3 andH4. Stable HeLa cell lines expressing histones H2B, H3, and H4 tagged withgreen fluorescent protein (GFP) were established; the tagged moleculeswere assembled into nucleosomes. Although H2B-GFP was distributed likeDNA, H3-GFP and H4-GFP were concentrated in euchromatin during interphaseand in R-bands in mitotic chromosomes. These differences probably resultfrom an unregulated production of tagged histones and differences inexchange. In both single cells and heterokaryons, photobleaching revealedthat H2B-GFP exchanged more rapidly than H3-GFP and H4-GFP. About 3% ofH2B exchanged within minutes, whereas approximately 40% did so slowly(t(1/2) approximately 130 min). The rapidly exchanging fractiondisappeared in 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole and so mayrepresent H2B in transcriptionally active chromatin. The slowly exchangingfraction was probably associated with chromatin domains surrounding activeunits. H3-GFP and H4-GFP were assembled into chromatin when DNA wasreplicated, and then >80% remained bound permanently. These results revealthat the inner core of the nucleosome is very stable, whereas H2B on thesurface of active nucleosomes exchanges continually.

The TATA sequence of the human, estrogen-responsive pS2 promoter iscomplexed in vivo with a rotationally and translationally positionednucleosome (NUC T). Using a chromatin immunoprecipitation assay, wedemonstrate that TATA binding protein (TBP) does not detectably interactwith this genomic binding site in MCF-7 cells in the absence oftranscriptional stimuli. Estrogen stimulation of these cells results inhyperacetylation of both histones H3 and H4 within the pS2 chromatinencompassing NUC T and the TATA sequence. Concurrently, TBP becomesassociated with the pS2 promoter region. The relationship between histonehyperacetylation and the binding of TBP was assayed in vitro using an invivo-assembled nucleosomal array over the pS2 promoter. With chromatin inits basal state, the binding of TBP to the pS2 TATA sequence at the edgeof NUC T was severely restricted, consistent with our in vivo data.Acetylation of the core histones facilitated the binding of TBP to thisnucleosomal TATA sequence. Therefore, we demonstrate that one specific,functional consequence of induced histone acetylation at a native promoteris the alleviation of nucleosome-mediated repression of the binding ofTBP. Our data support a fundamental role for histone acetylation atgenomic promoters in transcriptional activation by nuclear receptors andprovide a general mechanism for rapid and reversible transcriptionalactivation from a chromatin template.

Histone H2A.Z has a conserved function that is distinct from that of themajor H2A sequence variants.

Nucleic Acids Res. 2000; 28: 3811-6

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Saccharomyces cerevisiae contains three genes that encode members of thehistone H2A gene family. The last of these to be discovered, HTZ1 (alsoknown as HTA3), encodes a member of the highly conserved H2A.Z class ofhistones. Little is known about how its in vivo function compares withthat of the better studied genes (HTA1 and HTA2) encoding the two majorH2As. We show here that, while the HTZ1 gene encoding H2A.Z is notessential in budding yeast, its disruption results in slow growth andformamide sensitivity. Using plasmid shuffle experiments, we show that themajor H2A genes cannot provide the function of HTZ1 and the HTZ1 genecannot provide the essential function of the genes encoding the majorH2As. We also demonstrate for the first time that H2A.Z genes arefunctionally conserved by showing that the gene encoding the H2A.Z variantof the ciliated protozoan TETRAHYMENA: thermophila is able to rescue thephenotypes associated with disruption of the yeast HTZ1 gene. Thus, thefunctions of H2A.Z are distinct from those of the major H2As and arehighly conserved.

On the basis of a structural analysis of 240 protein-DNA complexescontained in the Protein Data Bank (PDB), we have classified theDNA-binding proteins involved into eight different structural/functionalgroups, which are further classified into 54 structural families. Here wepresent this classification and review the functions, structures andbinding interactions of these protein-DNA complexes.

The human TFIID components TAF(II)135 and TAF(II)20 and the yeast SAGAcomponents ADA1 and TAF(II)68 heterodimerize to form histone-like pairs.

Mol Cell Biol. 2000; 20: 340-51

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It has been previously proposed that the transcription complexes TFIID andSAGA comprise a histone octamer-like substructure formed from aheterotetramer of H4-like human hTAF(II)80 (or its Drosophila melanogasterdTAF(II)60 and yeast [Saccharomyces cerevisiae] yTAF(II)60 homologues) andH3-like hTAF(II)31 (dTAF(II)40 and yTAF(II)17) along with two homodimersof H2B-like hTAF(II)20 (dTAF(II)30alpha and yTAF(II)61/68). However, ithas not been formally shown that hTAF(II)20 heterodimerizes via itshistone fold. By two-hybrid analysis with yeast and biochemicalcharacterization of complexes formed by coexpression in Escherichia coli,we showed that hTAF(II)20 does not homodimerize but heterodimerizes withhTAF(II)135. Heterodimerization requires the alpha2 and alpha3 helices ofthe hTAF(II)20 histone fold and is abolished by mutations in thehydrophobic face of the hTAF(II)20 alpha2 helix. Interaction withhTAF(II)20 requires a domain of hTAF(II)135 which shows sequence homologyto H2A. This domain also shows homology to the yeast SAGA component ADA1,and we show that yADA1 heterodimerizes with the histone fold region ofyTAF(II)61/68, the yeast hTAF(II)20 homologue. These results areindicative of a histone fold type of interaction betweenhTAF(II)20-hTAF(II)135 and yTAF(II)68-yADA1, which therefore constitutenovel histone-like pairs in the TFIID and SAGA complexes.

Trypanosoma brucei is an extracellular protozoan parasite that cyclesbetween mammalian hosts and the tsetse vector. In bloodstream-formtrypanosomes, only one variant surface glycoprotein gene (VSG) expressionsite (ES) is active at any time. Transcriptional switching between ESsresults in antigenic variation. No VSG is transcribed in the insectprocyclic stage. We have used bacteriophage T7 RNA polymerase (T7RNAP) tostudy the transcriptional accessibility of ES chromatin in vivo. We showthat T7RNAP-mediated transcription from chromosomally integrated T7promoters is repressed along the entire length of the ES in the procyclicform, but not in the bloodstream form, suggesting that the accessiblechromatin of inactive bloodstream-form ESs is remodeled upondifferentiation to yield a structure that is no longer permissive forT7RNAP-mediated transcription. In the bloodstream form, replacing theactive ES promoter with a T7 promoter, which is incapable of sustaininghigh-level transcription of the entire ES, prompts an ES switch. Thesedata suggest two distinct mechanisms for ES regulation: achromatin-mediated developmental silencing of the ES in the procyclic formand a rapid coupled mechanism for ES activation and inactivation in thebloodstream form.

Sth1p, a Saccharomyces cerevisiae Snf2p/Swi2p homolog, is an essentialATPase in RSC and differs from Snf/Swi in its interactions with histonesand chromatin-associated proteins.

Genetics. 1998; 150: 987-1005

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The essential Sth1p is the protein most closely related to the conservedSnf2p/Swi2p in Saccharomyces cerevisiae. Sth1p purified from yeast has aDNA-stimulated ATPase activity required for its function in vivo. Thefinding that Sth1p is a component of a multiprotein complex capable ofATP-dependent remodeling of the structure of chromatin (RSC) in vitro,suggests that it provides RSC with ATP hydrolysis activity. Three sth1temperature-sensitive mutations map to the highly conservedATPase/helicase domain and have cell cycle and non-cell cycle phenotypes,suggesting multiple essential roles for Sth1p. The Sth1p bromodomain isrequired for wild-type function; deletion mutants lacking portions of thisregion are thermosensitive and arrest with highly elongated buds and 2CDNA content, indicating perturbation of a unique function. The pleiotropicgrowth defects of sth1-ts mutants imply a requirement for Sth1p in ageneral cellular process that affects several metabolic pathways.Significantly, an sth1-ts allele is synthetically sick or lethal withpreviously identified mutations in histones and chromatin assembly genesthat suppress snf/swi, suggesting that RSC interacts differently withchromatin than Snf/Swi. These results provide a framework forunderstanding the ATP-dependent RSC function in modeling chromatin and itsconnection to the cell cycle.

Recombinant yeast nucleosome assembly protein (yNAP-1) facilitates theformation of uniformly spaced nucleosomes from high molecular weight DNAand core histone octamers. No additional factors or metabolites arerequired. The repeat length of the chromatin produced is about 146 basepairs. To obtain the most distinct nucleosomal ladders, the core histonesmust preexist as an octamer complex. yNAP-1 forms complexes with corehistones as judged by native gel electrophoresis, chemical cross-linking,limited histone proteolysis, and affinity blotting. A discrete complex wasobserved with a probable ratio of yNAP-1 to histone octamer of 4:1.Chromatin produced by salt dialysis does not contain uniformly spacednucleosomes, but subsequent incubation with yNAP-1 creates uniformspacing. Trypsin-treated core octamers that lack amino termini, althoughcapable of forming core particles with core-length DNA by salt dialysis,are not assembled by yNAP-1 into uniformly spaced nucleosomes on highmolecular weight DNA. Proteolytic removal of the amino termini of the corehistones precludes complex formation between a histone octamer and yNAP-1.Affinity blotting also demonstrates that yNAP-1 binds linker histones andhigh mobility group (HMG)-1/HMG-2 but not HMG-14. Competition experimentswith poly-L-arginine, poly-L-lysine, and protamine reveal that yNAP-1binds to core and linker histones more tightly despite the much higherpositive charge densities of the former molecules. Naturally occurringacetylated histone H4 species show no evidence for differential yNAP-1binding. yNAP-1 is not bound tightly to the resulting chromatin afterdeposition and thus could act catalytically.

The four core histone proteins, H2A, H2B, H3, and H4 of Xenopus laevishave been individually expressed in milligram quantities in Escherichiacoli. The full-length proteins and the "trypsin-resistant" globulardomains were purified under denaturing conditions and folded into histoneoctamers. Both intact and truncated recombinant octamers, as well aschicken erythrocyte octamer, were assembled into nucleosome core particlesusing a 146 bp defined-sequence DNA fragment from a 5 S RNA gene. Thethree types of core particles were characterized and compared by gelelectrophoresis, DNase I cleavage, and tyrosine fluorescence emissionduring stepwise dissociation with increasing ionic strength. Nucleosomecore particles containing native and mutant histones made in bacteria havefacilitated its X-ray structure determination at 2.8 A resolution.

[Structure of chromatin. I: Levels of DNA organization in the nucleus;nucleosome and chromatin fibres].

Pathol Biol (Paris). 1994; 42: 868-83

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This bibliographic review, the first of a series of three on chromatinstructure, discusses the current knowledge and hypotheses concerningchromatin organization in nucleosomes and fibers. Presently, thestructural features of the chromatin arrangement are better known for thelow levels of organization, e.g. nucleosome, but there is muchdisagreement about levels of organization higher than the 10 nm fiber. Thepublished reports are consistent with the model that, in the nucleosome,the DNA is wrapped around the histone octamer. The histone octamer whosestructure and dimensions were investigated for years is viewed today as atripartite particle with the (H3-H4)2 tetramer flanked by two H2A-H2Bdimers. In the chromatosome, two turns of DNA around of the histoneoctamer are sealed by the histone H1 that binds DNA at the points of entryand exit. A chain of nucleosomes, each with about 200 bp associated DNA,forms a 10 nm fiber with a DNA packing ratio of 6-7. The 10 nm fiber isthen condensed into a 30 nm fiber, likely of solenoid structure, that isstabilized by histone H1. All the different models proposed for thestructure of the 30 nm fiber are consistent with a DNA packing ratio of35-40.

Synthesis of sperm and late histone cDNAs of the sea urchin with a primercomplementary to the conserved 3' terminal palindrome: evidence fortissue-specific and more general histone gene variants.

Proc Natl Acad Sci U S A. 1985; 82: 5676-80

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We have cloned histone cDNAs from total RNA isolated from testis and fromgastrula-stage embryos of the sea urchin Psammechinus miliaris. Thereverse transcription of histone mRNAs was specifically primed with anoligonucleotide that is complementary to the conserved palindromicsequence present at the 3' end of nonpolyadenylated histone mRNAs. Twosperm H2B, two late H2B, and three late H2A variant cDNA clones wereisolated and characterized by DNA sequence analysis. These cDNA cloneswere used to study the accumulation of histone mRNA during sea urchinembryogenesis. The different late H2A and H2B mRNAs are present in as fewas 200 copies in the egg and each accumulate to 3-5 X 10(5) molecules inthe gastrula embryo. One of the late mRNAs, the H2A-3 mRNA, is alsoabundant in testis RNA and codes for the H2A variant present in spermchromatin. The late H2A-3 protein is therefore a more prevalent H2Avariant of the sea urchin. In contrast, the two sperm H2B mRNAs are foundin testes but not ovaries and embryos of the sea urchin, suggesting thatthe sperm H2B genes are expressed only during spermatogenesis. Inaddition, evidence for gene conversion between two late H2A gene variantsis presented.

The process of dimer (H2A-H2B), tetramer (H3-H4)2 and octamer(H3-H4-H2A-H2B)2 formation was analyzed by the methods of gel-filtrationand differential spectrophotometry. Histone octamer with the parametersmost close to the native ones is established to be reconstructed only fromdenaturated monomers at neutral pH. The necessary condition for formationof histone octamer is the stage of tetramer (H3-H4)2 and (H2A-H2B)formation. Histone H3 with intramolecular disulphide bond (formula: seetext) does not form tetramer (H3-H4)2 and is not a part of octamer. Whenpassing through the dimer (H3-H4) stage it produces nonspecifichigh-molecular aggregates. Structural changes preceding the formation ofhistone octamer may play a significant role for transition of chromatinand nucleosomes into the transcription-active, repressed and other states.

When HeLa cells are lysed in solutions containing a non-ionic detergentand 2 M-NaCl, structures are released that retain many of themorphological features of nuclei. These nucleoids contain all the nuclearRNA and DNA but few of the proteins characteristic of chromatin. Their DNAis supercoiled and so intact. Using a simple and rapid procedure we havereconstructed nucleohistone complexes from nucleoids and the 'core'histones without breaking the DNA. We have probed the integrity andstructure of the reconstructed complexes using a non-destructivefluorometric approach, which provides a general method for detectingagents that bind to DNA and alter its supercoiling. The superhelicalstatus of the DNA in the reconstructed complexes is indistinguishable fromthat found in control nucleoids containing core histones. Experiments withmicrococcal nuclease confirm that the DNA in the reconstructed complexesis organized into nucleosome-like structures. These, however, are spaced145 base-pairs apart and not 200 base-pairs apart as is found in nativechromatin.

The roles of H1, the histone core and DNA length in the unfolding ofnucleosomes at low ionic strength.

Nucleic Acids Res. 1980; 8: 4969-87

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Calf thymus nucleosomes exhibit two different and independent hydrodynamicresponses to diminishing salt concentration. One change is gradual overthe range 40 to 0.2 mM Na+ and is accompanied by decreases in contact-sitecross-linking efficiency. The other change is abrupt, being centeredbetween 1 and 2 mM Na+. We found only one abrupt change in sedimentationrate for particles ranging in DNA content fom 144 to 230 base pairs. Thisresponse to decreasing ionic strength is similar for particles of both 169and 230 base pairs. Core particles (144 base pairs) exhibit a somewhatdiminished response. The abrupt change is blocked by formaldehyde ordimethylsuberimidate cross-linking. The blockage by dimethylsuberimidatedemonstrates that the abrupt conformational change requires theparticipation of the core histones. H1 completely blocks the abrupt butnot the gradual conformational change. Thus H1 uncouples the differentresponses to low ionic strength and exerts an important constraint on theconformational states available to the nucleosome core.

Binding of cis- and trans-dichlorodiammineplatinum(II) to the nucleosomecore.

Proc Natl Acad Sci U S A. 1979; 76: 6091-5

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The binding of the antitumor drug cis-dichlorodiammineplatinum(II) and itsinactive trans isomer with the nucleosome core particle has beeninvestigated. Kinetic studies show that platinum binding increases withincubation time, from a few bound platinum atoms per nucleosome core inthe first 0.5 hr to 40-50 after 40 hr. There is no crosslinking ordissociation of nucleosome cores upon platinum binding, as revealed bysedimentation velocity measurements. Electrophoresis through 0.1% sodiumdodecyl sulfate/18% polyacrylamide gels after platinum binding revealsstriking differences in the DNA and protein band patterns for the twoisomers. cis-Dichlorodiammineplatinum(II) binds first to the DNA,retarding and spreading its migration in the gel. A comparison study withthe 146-base-pair nucleosome core DNA showed the binding to be littleaffected by the presence of the histone octamer. The trans complex, on theother hand, produces DNA-histone and histone-histone crosslinks that onlyappear for the cis isomer after long incubation times. The protein-proteincrosslinks were reversed by soaking the gel in cyanide solution to form[Pt(CN)(4)](-2). Subsequent two-dimensional gel electrophoresis revealedthat trans-dichlorodiammineplatinum(II) forms specific crosslinks betweenhistone protein pairs H3 and H2a and H2b and H4 in the nucleosome core.The occurrence of DNA-protein crosslinks was demonstrated by treating theplatinum/nucleosome core reaction mixtures with a protease or withnucleases prior to electrophoresis and observing changes in the gelpatterns. Platinum was located in the gels through autoradiography using(195m)Pt-labeled complexes. This work clearly demonstrates the greaterpropensity of trans-dichlorodiammineplatinum(II) to form histone-histoneand histone-DNA crosslinks compared with the antitumor active cis isomer,which binds first to the DNA and only forms crosslinks to the histoneswhen the nucleosome core is heavily loaded with platinum.